Method and apparatus for accelerating biotechnical reaction and production

ABSTRACT

The method and apparatus according to the present invention concentrate the formation of the biotechnical product to active site in the reactor, where the production is accelerated to extreme speeds by altering the conditions. To achieve this, anabolic, catabolic or overflow metabolic reactions can be utilised. The aim is to implement production so that carbon dioxide emissions in particular are minimised The products are e.g. 2,3-butanediol, butanol, ethanol, acetone, organic acids, methane or hydrogen gas and other fuels or compounds necessary for chemical or material production.

BACKGROUND

Biotechnological production processes, used to produce fuels and chemicals from organic starting materials, biomasses, are called biorefinement. Today's oil refineries, in fact, are based on the chemical methods, which were in use for biomass processing since the 1800s. Also involved is the strong development of microbiology starting in the mid-1800s, which has significantly contributed to our understanding of, among other things, naturally occurring degradation processes; they occur because of living organisms invisible to the naked eye. Some of these organisms are able to convert biomass degradation products to economically useful products, which can not only be utilized for energy production, but also as feedstock in various industrial chemical processes.

There have been extended periods, particularly after World War II during which crude oil and other fossil fuels were relatively inexpensive. The so-called petrochemical industry, which produces the chemical fractions that act as fuel and feedstock for other processes, is built upon these inexpensive raw materials. Burning fossil fuels has in turn led to environmentally damaging developments, such as acid rain, greenhouse gases, and possibly climate change. Energy crises have shown the vulnerability of the crude oil based economic system in situations where the price of oil has rapidly increased, or the availability of fossil fuels has otherwise been compromised by, for example, crises or varying political agendas. It is therefore very prudent to develop bioprocess solutions, based on inexpensive biomass such as waste materials. The processing of waste materials also contributes to the formation of environmental protection solutions within the economic system. The waste used can also be gaseous.

The metabolic reactions of micro-organisms essentially often release chemical energy from organic material. For this reason, the external energy consumption of microbial processes is usually low and the energy balance positive. This, and the fact that with biotechnical processes the same equipment can be used to carry out different reactions from variable raw materials, should be utilized as well as possible. Likewise, the thermal energy in gases can be utilized by committing it to the process. Similarly, the compounds in the gas can be made to react with the compounds in the process bath. This type of reaction can occur catalyzed by microbes and their enzymes. At the same time the environmental load of emissions from combustion plants are reduced.

Microbial metabolic reactions are mainly anabolic, related to the construction of cells, and catabolic i.e. cell energy usage. In some circumstances, microbes are content to just process the molecules in the environment, in order to change the conditions to be beneficial or to keep existing beneficial circumstances in place. These reactions may also contain symbiotic interactions between microbes. Sometimes bacterial cells consume more chemical energy than they need. The energy is then either stored in the cells, or bound to the large quantity of synthesized product without causing significant cell growth (cell division). This is often referred to as overflow metabolism.

Overflow metabolism is a verified phenomenon in the metabolism of many bacteria. It's not really a catabolic or an anabolic reaction. Bacteria using overflow metabolism neither use the chemical energy released in this reaction for their own needs, nor build new cell growth on this metabolism. In terms of the bacterial population the main purpose of overflow metabolism is to change the environment so that the conditions necessary for life are maintained or improved. Overflow metabolism occurs especially when abundant carbon and energy sources are available. Through this action, bacteria are trying to compete with other microbes, e.g. by consuming the extra nutrients so that they're out of the reach of competitors.

Since overflow metabolism is not related to bacterial growth, its regulatory mechanisms may differ from normal metabolic regulation, and remain largely unknown. The research has also been limited by the fact that, in terms of bacteria, they often represent extreme or atypical conditions. These reactions clearly play an important part in the circulation of matter in nature as a part of microbial metabolism. Thus, these reactions can be made use of industrially, for example in fuel or chemical production. Similarly they can be used to convert gaseous compounds to a liquid or solid phase.

Microbial metabolism is usually divided into energy-producing respiratory or fermentation (catabolic), and anabolic cell building-related reactions. At the cellular level, these reactions usually occur simultaneously. Additionally, microbial survival under different conditions may also be associated with other reactions, such as the emergence of highly durable forms (eg, bacterial spores) or overflow metabolism. The purpose of these reactions is to enable the survival of the bacterial population. Their industrial exploitation has not yet been specifically explored, although they may involve significantly rapid substance transitions or reactions.

When the microbial population is introduced to favorable circumstances, it generally moves to the active growth phase (exponential growth). It is preceded by the so-called lag phase, during which the population cells synthesise the enzymes and other molecules necessary for growth, and are thus “restocked.” Growth stage may be a multi-tiered, due, for example, to the tendency of microbes to first use up one main carbon and energy source from their substrate, and then move on to other sources. (e.g. secondary metabolism). Often this requires the synthesis of new enzymes, including hydrolytic.

Under natural conditions (non laboratory), microbes often occur in mixed populations. Their activities involve various interactions, such as commensalism. As an example, the pH control of the beginning of the small intestines has been linked to the co-operation of facultatively anaerobic bacteria (Hakalehto, 2008). Changes in gut flora will also affect the life of the host organism. Concerning obesity, it has been established that individuals with a tendency towards obesity have a greater concentration of butyrate producing bacteria in the first part of the colon.

For example, 2,3-butanediol production takes place in the intestinal tract in a much earlier stag; namely in the duodenum and the rest of the small intestine. The human body absorbs 80% of its nutrients in those regions. Rapid use of nutrients by the host as well as by the microbial flora is particularly fast, and therefore thick biofilms, such as in the colon, cannot form on the intestinal walls. Other anaerobic metabolic reactions include, for example: acetone-butanol fermentation, ethanol fermentation, methane fermentation and production of several organic acids.

The bacteria active within flora of the beginning of the small intestines, including the duodenum, consist particularly of those being highly bile resistant and commensalistically capable, even in those circumstances (Hakalehto et al, 2010). Additionally, under these circumstances because of the actions of, among others, Klebsiella sp. and Enterobacter sp. genera of bacteria, ethanol and 2,3-butanediol are formed. Thus, by exploring these intestinal conditions it is possible to learn useful things pertaining to the industrial production of these chemicals. The important thing is that the active microbes collaborate with many other microbial components, such as the Escherichia coli. A method for efficient culturing of these bacteria is presented in the Example 1. The pH drop caused by the organic acids produced by E. coli and other bacteria carrying out mixed acid fermentation is balanced by the neutral components, ethanol and 2,3 butanediol, produced by Klebsiella and Enterobacter group of bacteria. In all of these and most other bacterial metabolic reactions differing amounts of carbon dioxide, CO₂, are released. If the carbon dioxide that was produced in the reaction or otherwise introduced to the process can be made to react or otherwise combine with the process bath, the amount of CO₂ released is reduced. If the CO₂ or carbon monoxide is a product of combustion or incineration and its release into the atmosphere when introduced to the bio-process is reduced as described above, then the general environmental burden and environmental impact of the specific combustion or incineration is lessened.

DESCRIPTION OF THE INVENTION

With the invented method and apparatus it is possible to:

-   -   1. Transform multiple biomass based waste materials to valuable         raw materials enabling their recycling.     -   2. Reduce the greenhouse gas emissions of manufacturing         biotechnical chemicals and fuels.     -   3. Bind the carbon molecules present in biomass to products and         commodities such as durable plastic or rubber-based materials         (if they are not consumer goods the carbon emissions can be         bound to the material for a long time).     -   4. Utilize the chemical energy released from waste without a net         increase in CO₂ emissions in energy production. Also, the         released CO₂ can be bound to a under glass plant or algae         material (tissue).

In the light of the aforementioned benefits it is easy to see how a method using this invention can significantly increase the ecological efficiency of the chemical industry and energy production. This method according to the present invention and the device based on it can be used for producing gasoline equivalent butanol, or ethanol as a fuel for internal combustion engines.

Similarly, 2,3-butanediol, acetone, organic acids or other compounds can be produced as raw materials for the chemical industry or gases such as methane or hydrogen, for energy production. At the same time one product of the process is reduced organic waste and environmental load. As seen in the Example 2, the volume of gases like CO₂ can be reduced using methods detailed by this invention and consequently the amount of greenhouse gases is reduced. In this way carbon can be bound to different plastics or synthetic rubbers, which can be used in various long lived purposes like for road surface materials.

A common problem with biotechnical reactions are diffusion limitations. This means that substances necessary for microbe nutrition or product formation do not reach the microbes or other biocatalysts quickly enough, and respectively waste products don't leave fast enough. With the presently invented method and apparatus gas is added to the active site to speed up diffusion. The diffusion problem is lessened due to the sped up production reaction or its main parts taking place within the limited space of a small part of the reactor. In this way, the physical conditions such as temperature or pH, can be adjusted for optimal product formation. Bioreactor itself can be implemented with a sectoral basis (FIGS. 1 and 2), or in the shape of the pool or with belt (FIG. 3).

All the reactions taking place in bioreactor can be measured and regulated through the central unit, which in turn can be controlled remotely. In this way, the bioreactor can operate automatically but is still under constant surveillance.

In order to obtain ideal diffusion conditions for the formation of a biotechnical product the invented method and apparatus injects the heart of bioreactor (“active site”) with the necessary additions. These may include gas flow, or other additions maintaining favorable conditions, such as by injecting gaseous substances to sustain optimal production temperature in order to create ideal reaction conditions. Liquids or suspensions, may also be added. Added substances or materials may be different nutrients, growth factors, raw materials, regulatory factors, microbial cultures or any other substrates or microbes useful for production. In this patent application, substrate means a gas, liquid or solid substance added to the reaction, reaction broth and the active site which takes part in the reaction or its regulation. Reaction broth or process bath, or broth stands for the entire main portion of the substances taking part in the reaction, which can be inoculated with production microbes or enzymes, or it can be uninoculated. The bath or broth may contain the naturally occurring microbes of its biomass raw materials.

At this point it's usually possible raise the flow speed of the bath or broth quite high due to high productivity. Similarly, pretreatments, such as enzymatic or other hydrolytic reactions or microbial growth, are slower and so call for slower flow speeds and higher volumes. One of the core ideas of this present invented method and apparatus, is to concentrate the conditions necessary for high reaction speed into the heart of the bioreactor. This may include, for example, pH adjustment, as well as the adding in of various nutrients and regulatory substances, or other substances. Various control measures may also be implemented through the addition of gaseous substances. for example, the beginning of Escherichia coli growth, can be accelerated by adding carbon dioxide (Hakalehto, 2011). If combustion fumes containing carbon dioxide and/or carbon monoxide are led into the bioreactor, these gases and the carbon they contain can contribute to the formation of reaction product, as shown in Example 2. The carbon sequestration can then be used to reduce the climate impact of the combustion gases and to restore carbon bound in the bioreactor to a liquid and/or solid state.

In the same way as in the different phases of biomass pretreatment or microbe propagation i.e. culturing, the after-treatment can also include the handling of growing volumes when compared to the production reaction action site in the core of the bioreactor. The speed of the process can also be adjusted by adjusting the flow rate. Not only the reaction bath flow, but also the flow rate of different substance additions and the pH and temperature can be regulated. To facilitate adjustments it is essential that chemical sensors be installed in varying locations in the bioreactor to measure physical or biological parameters.

The bioreactor can be implemented in a flow pool with depth variations (FIG. 3). Then in both cases, the reactor volume is at its lowest in the bioreactor core in (x), and it is to this point that the necessary additions fort accelerating the reaction are added. These may include microbe addition. All additions can be achieved, for example by bubbling, spraying, injecting, pouring, or other equivalent means. Pre-treatment is carried out in Part A, after-treatment in Part B. The corresponding markings have been made to the figure where the reactors A- and B-parts are both sector-shaped. The total volume can be adjusted by moving the sector side walls, and thus enlarging the scope of sector or a reduction. Basin-shaped reactor volume can be adjusted by changing the height of the running belt. Flow basin and the belt can of course be placed in a tube. In this case, biotechnical process and production can be either partially or fully implement during the process-, i.e. reaction-bath transport.

Example 1 Growing Bacteria in a PMEU Device (Portable Microbe Enrichment Unit)

Inoculating bacterial culture propagated overnight in THG-nutrient broth tubes (tryptone-yeast extract-glucose) to the corresponding substrate in a PMEU-cultivation syringe. This is transferred to a PMEU (Portable Microbe Enrichment Unit) growing device suitable growth temperature. With E. coli and K. mobilis—bacteria, this temperature may be +35 or +37° C. Culturing is carried out as described previously: aerobically, anaerobically or microaerobically (Hakalehto et al, 2008).

Example 2

Klebsiella mobilis and Escherichia coli mixed cultures in microaerobic conditions of +37° C. yielded two isomers of 2,3-butanediol, as seen in Table 1. K-culture is grown in a THG (tryptone-yeast extract-glucose) substrate containing 20% enzymatically hydrolysed sawdust, while the KS cultures also contained 10% of enzymatically hydrolysed cellulose. Acetate (0.1%) was added to 9.5 hours after incubation and additional THG substrate after 11.5 hours, when the pH was adjusted to 6.5. Microbial cultures were added at the same time point (5% K. mobilis and 5% of e-coli culture), and 3 ml cellulase enzyme solution. Container volume was 8 l, which reflects the bioreactor production points, i.e. the points to which the necessary additions are made. When the incubation had lasted 36 and 47 hours, 4 ml and 3 ml, respectively, of cellulase enzyme was added. K-cultures began an intense gas formation at 17.5 hours time, which gas presumably consisted mainly of carbon dioxide. However, bubbling, was almost non-existent in the KS culture, which contained more cellulose derived glucose (197 mg/dl in the beginning of the reaction versus the 36 mg/dl glucose levels of the K-culture). Thus, the overflow metabolism realised in the KS-culture resulted in increased production of 2,3-butanediol, which can bee seen in Table 1, without high gas e.g. carbon dioxide production or emission. The fact that this is mainly overflow metabolism is seen in Table 2. It is assumed that carbon dioxide formed in bacterial respiration, or the fermentative reactions in KS culture, was utilized as a part of the cultures metabolism.

LITERATURE

-   E. Hakalehto, T. Humppi, H. Paakkanen, Dualistic acidic and neutral     glucose fermentation balance in small intestine: Simulation in     vitro. Pathophysiology (2008) -   E. Hakalehto, M. Hell, C. Bernhofer, A. Heitto, J. Pesola, T.     Humppi, H. Paakkanen, Growth and gaseous emissions of pure and mixed     small intestinal bacterial cultures in PMEU: Effects of bile and     vancomycin. Pathophysiology (2010) -   E. Hakalehto, Simulation of enhanced growth and metabolism of     intestinal Echerichia coli in the Portable Microbe Enrichment Unit     (PMEU). M. C. Rogers and N. D. Peterson (Eds.), E. coli     interactions: causes, treatment and prevention. Nova Publishers, New     York, USA. In Press.

TABLE 1 The amount of 2,3-butanediol formed by the mixed culture of Klebsiella mobilis and Escherichia coli (K) in proportion to the butanediol production levels in the KS-culture as percentages. Description of the conditions in the text part of the Example 2. The elevated butanediol production is combined with reduced gas emissions in the reaction. KS/2,3- K/2,3- KS/2,3- K/2,3- 2,3- Time point butanediol butanediol butanediol butanediol butanediol (h) isomer 1 isomer 1 isomer 2 isomer 2 (K/KS) 0 0 0 0 18.20 — 4.5 57.43 0 29.31 18.65 21.5% 7.5 67.71 0 56.72 21.74 17.5% 10.5 56.34 0 58.59 43.26 38.8% 14.5 0 57.88 46.26 40.81 213.3% 16.5 55.51 59.54 197.11 43.31 40.7% 17.5 76.92 57.01 90.14 52.66 66.4% 21.5 94.15 60.27 108.98 57.56 58.0% 24.5 101.46 66.72 99.93 48.24 57.1% 27 133.84 76.17 94.46 53.22 56.7% 28 195.89 70.58 130.16 43.79 35.1% 29.5 213.99 99.86 182.53 63.59 41.2% 34 248.99 109.44 135.74 57.52 50.9% 46.5 289.53 147.76 147.58 54.11 46.2% 49.5 248.05 152.16 145.26 45.66 50.3% 51.5 326.95 99.16 204.75 53.38 28.7% 54 345.36 129.70 174.53 53.34 35.2%

TABLE 2 The bacterial concentrations of the K- and KS cultures (cfu, colony forming units) in a milliliter at time point 253 hours, when they contained 10-fold difference as determined by TYG-agar. This implies to increased 2,3-butanediol production being not related to the anabolic reactions in the cultures but to the overflow metabolism. bacterial concentration Culture K KS E. coli 3 × 10 exp 7 2.4 × 10 exp 8 K. mobilis 6 × 10 exp 7 2.5 10 exp 8 

1. A biotechnical production method, said method comprising the steps of: creating ideal conditions that take place in at least one location within a core of a bioreactor, in which said location a reaction occurs is as close to an optimal level as possible and despite a rapid flow of a reaction bath a high productivity achieved through said conditions enables abundant product formation.
 2. The method in accordance with claim 1 further comprising the step of introducing of a gas or a mix of gases beneficial to the production process to said location, even if said conditions in other parts of said reactor are different.
 3. The method in accordance with claim 2, wherein said gas or said mix of gases contains at least one of carbon dioxide, carbon dioxide and monoxide, and carbon monoxide, that binds in part with said reaction bath after being formed in a combustion reaction, which binding to a bioreaction lessens environmental load and climate impact of combustion reaction's gas emissions.
 4. The in accordance with claim 3, further comprising the step of introducing nutrients, growth factors, precursors, regulatory factors, microbial cultures or other substrates or microbes being beneficial to the formation of, or the speeding up of the production of, the product.
 5. The method in accordance with claim 4 wherein said reaction carried out by the microbes is an overflow reaction, which does not primarily serve the growth of the microbe or the energy production for the needs of acute cell metabolism.
 6. The method in accordance with claim 5, wherein the microbes carrying out the reaction produce ethanol, 2,3-butanediol or other organic chemicals exploitable industrially or as a fuels, or hydrogen, methane or other gases suitable for energy production.
 7. The method in accordance with claim 6, wherein the reaction there are formed lower carbon dioxide emissions when compared to metabolic reactions of the microbe in question, or to the gas flow introduced to the substrate.
 8. An apparatus for biotechnical production, said apparatus comprising: a circular or sectoral basin configured to allow a flow to travel towards center (p) of a radius (r) while getting also faster, said basin being configured to receive an amount of raw material.
 9. The apparatus in accordance with claim 8, wherein at least one location of active sites of a production reaction is near said center.
 10. The apparatus in accordance with claim 9, wherein an exit flow takes place from an opposite side of said active site to another sector, or vertically to another level above/below said active site.
 11. The apparatus in accordance with claim 10, wherein said basin has side walls that are moveable to alter a sector area and respectively a liquid volume of the reactor.
 12. The apparatus in accordance with claim 7, wherein a reaction broth or precursors travel with a flow or on a belt to and past said active site.
 13. The apparatus in accordance with claim 12, wherein said flow is placed within a tube.
 14. The apparatus in accordance with claim 13, wherein a gas, substrates, microbes or other substances are introduced to the active site by one of bubbling, spraying, injecting, and pouring.
 15. The apparatus in accordance with claim 14, wherein a volume of the reaction bath is altered based on controlling a channel depth in different locations, or through a level of a liquid on top of the belt.
 16. The apparatus in accordance with claim 15 further comprising sensors installed in varying locations in the bioreactor to measure chemical, physical or biological parameters, results of the measurements being used to control the introduction and/or rationing of the gases, substrates or microbes to the active site.
 17. The apparatus in accordance with claim 16, wherein the gas, substrate or microbe culture added to the active site is used to control the temperature, pH or equivalent reaction conditions.
 18. The apparatus in accordance with claim 17, wherein the bioreactor system is controlled through a central unit, which control can take place electronically or also wirelessly from long distances. 